Immune CD8 T cells are essential in cellular immunity against many intracellular pathogens including Listeria monocytogenes. In studying the host response to murine listeriosis, we have recently made several unexpected new observations which merit study: 1) By conventional rules for antigen processing, and exogenous antigen should not be processed by macrophage accessory cells into a form recognizable by CD8 cells. Yet heat-killed listeria are readily presented in this manner. We recently have identified a physiologic basis for this observation, namely a novel pathway which processes listerial antigens as an exogenous antigen and then shunts the processed fragments into the "endogenous" pathway for presentation to CD8 cells. In the future, we will use biochemical and molecular approaches to isolate the immunogenic listerial products and "antigen presenting molecules" associated with this atypical processing. We will also examine the importance of the pathway in presenting bacterial antigens in vivo. 2) Unlike other well defined antigens which can only be presented to T cells by syngeneic accessory cells, listerial antigens can be presented to T cells by a broad range of allogeneic accessory cells, a phenomenon known as MHC unrestricted recognition. Since CD8 T cell receptors (TCR) are a key element in recognition, we recently examined the V-beta regions expressed on listeria-immune CD8 cell lines and found an unusual, skewed pattern of usage. Based on the findings of others studying T cell recognition of bacterial products, we believe these receptors may bind to listerial products through a variant of the recently described "superantigen" phenomenon. In the future we will use immunological and molecular techniques to analyze TCR structure in a panel of epitope- specific CD8 clones. In this way, we hope to identify salient features associated with unrestricted recognition. 3) IFN-gamma is essential in host immunity to Listeria. Based on indirect evidence, it has previously been assumed that Listeria-immune T cells were the most important source of this vital factor during infection. Recent results in our lab using molecular techniques to directly monitor lymphokine mRNA levels indicate a) that NK cells are very active producers and b) T cells are quite poor producers of IFN-gamma mRNA in vivo. If IFN-gamma protein production follows similar patterns, prior concepts of the sources and the role of IFN in bacterial infection may need major revision. We propose to apply in situ hybridization and immunohistochemical techniques to monitor and compare IFN mRNA and protein production during infection. If our prior findings are corroborated, we will attempt to identify regulatory factors responsible for these surprising results. There is a strong possibility that additional studies pursuing our previous findings will provide major new insights into the recognition of bacterial pathogens by CD8 T cells and into the unique role of CD8 cells in host immunity. Ultimately, the insights obtained may be exploited in developing new strategies (using immunogenic peptides and recombinant cytokines) for enhancing CD8 cell function in particularly in patients with defects in T cell function.